SUMMARY An effective vaccination strategy is urgently needed to combat the global scourge of tuberculosis (TB). Due to the known importance of CD4 T cells and IFN? in immunity against TB, current vaccine efforts are focused on boosting bulk IFN?-producing CD4 T cell numbers (i.e., Th1 cells). These efforts, however, have been only moderately successful in conferring protection against TB in animal models, and a recent human efficacy trial of a novel candidate vaccine failed to confer measurable protection despite significantly boosting a Th1 response. Thus, there is growing concern that TB vaccines that target Th1 cells may not be adequately effective. We, and others, have recently discovered that adoptive transfer of fully differentiated Th1 cells, expressing high levels of the Th1-promoting transcription factor T-bet, provide little or no protection against murine TB, at least in part because they localize poorly to the lung parenchyma, the primary site of infection. In contrast, less-differentiated CD4 T cells, expressing intermediate levels of T-bet and sharing properties with Tfh and central memory T cells, readily home to the M. tuberculosis (Mtb)-infected lung parenchyma and mediate superior protection. Importantly, however, we have also recently shown that Mtb infection itself drives Th1 cells toward terminal differentiation and a non-protective state. This complicates vaccine approaches in regions of the world in which TB is endemic because most individuals who would benefit from immunization have already been exposed to Mtb or are persistently infected. The goal of this proposal is to elucidate the mechanisms that enable protective CD4 T cell populations to enter and be maintained within the lung parenchyma and to evaluate the influence of pre-existing Mtb infection on these parameters during vaccination in both animal models and human clinical trials. In Aim 1, we will determine the factors that govern the localization of CD4 T cells into the parenchyma, with the prime hypothesis that T cell KLRG1 binds N-cadherin on vascular endothelial cells, thus preventing entry. In addition, the role of key cytokines and glycolipid interactions in granuloma-associated high endothelial venules will also be explored. In Aim 2, we will assess whether vaccine-dependent CD103 expression serves to retain protective CD4 T cells within the parenchyma, and if so, whether this is beneficial or detrimental to immunity. Finally, in Aim 3, we will determine the impact of vaccine dose on priming protective, lung-homing CD4 T cell responses and how previous Mtb exposure influences this priming. The access to clinical samples from dose escalation studies of infected individuals provides a unique opportunity to compare human immune profiles to those observed in mice and to translate our murine findings into testable hypotheses for human TB. The proposed experiments will provide a framework for the future design of novel vaccine delivery strategies with the ability to induce and maintain lung- localizing T cells even in TB endemic regions.